The human genome contains thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability, or translation of their overlapping genes 1,2 . We describe MAPT-AS1, a primate-conserved, brain-enriched NAT containing an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing with rRNA pairing to MAPT mRNA internal ribosome entry site (IRES) 3 . Tau, a neuronal intrinsically disordered protein (IDP), stabilises axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies 4 . MAPT mutations cause familial frontotemporal dementia (FTLD-tau), and common variation forming the MAPT H1 haplotype is a significant risk factor in many tauopathies 5 , and Parkinson's disease. Notably, expression of MAPT-AS1 or its minimal essential sequences including MIR reduces, whereas silenced MAPT-AS1 increases neuronal tau, and is correlated with tau pathology in human brain. Moreover, we identified hundreds additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration, and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. Collectively, we present the importance of MAPT-AS1 for tauopathies, while also uncovering a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs 6 , with particular relevance for proteostasis in neurodegeneration.

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A wide variety of mammalian cell types is used in gene transfection studies. Establishing transfection methods that enable highly efficient DNA uptake has become increasingly important. PC12 is an established rat pheochromocytoma cell line, which responds to exposure to NGF with cessation of growth, expression of cytoplasmic processes, and differentiation into cells resembling sympathetic neurons. Although PC12 cells represent an important model system to study a variety of neuronal functions, they proved relatively difficult to transfect. We have compared the efficiency of three different chemical transfection reagents (Lipofectamine 2000, Lipofectamine LTX and TransIT-LT1) and of two electroporation systems (Neon and Gene Pulser Xcell) in transiently transfecting undifferentiated PC12 cells. By comparing efficiencies from replicate experiments we proved electroporation (in particular Neon) to be the method of choice. By optimizing different parameters (voltage, pulse width and number of pulses) we reached high efficiency of transfection (90 %) and viability (99 %). We also demonstrated that, upon electroporation, cells are not altered by the transfection and maintain their ability to differentiate.

Frontotemporal dementia (FTD) is a neurodegenerative disorder characterized by degeneration of the fronto temporal lobes and abnormal protein inclusions. It exhibits a broad clinicopathological spectrum and has been linked to mutations in seven different genes. We will provide a picture, which connects the products of these genes, albeit diverse in nature and function, in a network. Despite the paucity of information available for some of these genes, we believe that RNA processing and post-transcriptional regulation of gene expression might constitute a common theme in the network. Recent studies have unraveled the role of mutations affecting the functions of RNA binding proteins and regulation of microRNAs. This review will combine all the recent findings on genes involved in the pathogenesis of FTD, highlighting the importance of a common network of interactions in order to study and decipher the heterogeneous clinical manifestations associated with FTD. This approach could be helpful for the research of potential therapeutic strategies.

Nucleic acid therapeutics are witnessing an impressive acceleration in recent years. They work through multiple mechanisms of action, including downregulation of gene expression and modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in Microtubule-Associated Protein Tau (MAPT) exon 10 splicing reporter. We applied HCS-Splice to the wild-type and mutant reporters and measured the functional change in exon 10 inclusion. To demonstrate the applicability of the method to cell-based drug discovery, HCS-Splice was used to evaluate the efficacy of an exon 10-targeting siRNA, which was able to restore the correct alternative splicing balance.